Composition and method for the detection of uric acid

ABSTRACT

METHOD FOR DETECTING URIC ACID IN AQUEOUS FLUIDS WHICH COMPRISES CONTACTING THE FLUID WITH A COMPOSITION COMPRISING A FERRIC SALT AND EITHER 2,4,6-TRI(2-PYRIDYL)-1,3,5-TRIAZINE OR 2,2&#39;&#39;:6&#39;&#39;,2&#34;-TERPYRIDE, IN A BUFFERED ACIDIC MEDIUM. A PREFERABLE EMBODIMENT COMPRISES INCORPORATING THE COMPOSITION WITH A CARRIER AND USING THE RESULTING COMBINATION AS A DIP AND READ TEST DEVICE. A FURTHER PREFERABLE EMBODIMENT COMPRIESSES THE USE OF A PERSULFATE IN THE COMPOSITION WHICH INCREASES THE SPECIFICITY AND STABILITY OF THE COMPOSITION.

United States Patent O 3,711,252 COMPOSITION AND METHOD FOR THEDETECTION OF URIC ACID Ashim Chandra Roy, 62 Gloucester Drive, London,England No Drawing. Filed Oct. 13, 1971, Ser. No. 188,942 Claimspriority, application Great Britain, Oct. 28, 1970,

51,276/ 70 Int. Cl. C07d 57/34; C09k 3/00; Gln 33/16 US. Cl. 23-253 TP 9Claims ABSTRACT OF THE DISCLOSURE BACKGROUND OF THE INVENTION It is wellknown that the amount of uric acid found in the blood or urine iselevated when a person has gout, uremia, leukemia or pneumonia. Uricacid is normally present in blood serum in amounts varying from 0.7 to 6percent mg. but in the disease conditions stated, values of percent mg.or higher are common.

Medical science has long recognized the usefulness of a test for uricacid in blood serum or urine as an aid in diagnosing abnormal conditionssuch as gout and distinguishing it from arthritis: in gout the uric acidincreases whereas in arthritis no increase occurs. Several methods areknown for determining uric acid in body fluids, but no rapid, simple andreliable dip and read test for uric acid has heretofore been reported.

SUMMARY OF THE INVENTION The present invention provides compositions forthe detection of uric acid which comprise a ferric salt, a persulfateand either 2,4,6-tri(2-pyridyl)-1,3,5-triazine (hereinafter called TPTZ)or 2,2':6',2"-terpyridyl, in a buffered acidic medium.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Uric acid causes a color changein such above noted compositions which change is directly proportionalto the quantity of uric acid present. It is believed that ferric ironforms a complex with either TPTZ or terpyridyl which is reduced by uricacid to a ferrous complex thus causing a color change directlyproportional to the concentration of uric acid. The color changereaction may be carried out in aqueous or alcoholic solution or onprepared strips of absorbent material pretreated with the reagent underacid conditions. The addition of the persulfate, e.g. ammoniumpersulfate, increases the specificity of the reaction, probably bypreventing interference by ascorbic acid. It also increases thestability of the composition.

The color change reaction should be effected in an acidic medium of pH2-6, and preferably about 4. Such a pH can be maintained by the presenceof buffering agents such as glycinehydrochloric acid, glycine-aceticacid, sodium acetate-acetic acid, glycine-sulfamic acid or sodiumsuccinate-succinic acid mixtures.

Ferric chloride is the preferred ferric salt, but any other siutableorganic or other inorganic ferric salt may be em ice ployed, e.g. ferricsulfate, ferric alum, bromide, fluoride, nitrate, phosphate, acetate, oroxalate. The ferric salt is best employed in a ratio of one part byweight of said salt to 2 parts by weight of TPTZ or terpyridyl.

It is preferable to incorporate the test composition on or with acarrier member and to utilize this combination as a dip-and-read testdevice. This can be readily achieved, for example, by impregnating abibulous material with a solution of the test composition and thereafterdrying the impregnated test device. Alternatively, a finely divided,dry, intimate mixture of the ingredients can be adhesively attached tothe surface of the carrier. Another embodiment comprises incorporatingthe test composition with a semi-permeable polymeric membrane and usingthe combination as such or further attaching such membranes to acarrier. The carrier material of the present invention may be filterpaper, wood strips, synthetic plastic or non-woven or woven fabric.Filter paper having a thickness of from 0.01 inch to 0.02 inch ispreferred as the carrier.

Light interferes with the color change reaction. The compositions of theinvention should be stored in the dark, and the color change reactionperformed so far as practicable in the dark or at least in the absenceof strong light, especially sunlight.

The following examples illustrate the invention.

Example 1 One half m1. of a protein-free serum was mixed with 4 ml. of a0.1 molar glycine-HCl buflier (pH=3.8), 0.3 ml. of a solution containing2 mg. of TPTZ per ml. of ethanol, and 0.2 ml. of a solution containing1.5 mg. of ferric chloride and 10 mg. of ammonium persulfate per ml. ofethanol. The mixture was held in the dark for 15 minutes and theintensity of the blue color which developed was read at 593 millimicronsagainst a reagent blank in a spectrophotometer. The amount of uric acidpresent in the serum was obtained by reference to a calibration curveprepared at the same time using ethanol solutions containing knownamounts of uric acid.

Example 2 To 300 mg. of TPTZ dissolved in 2 ml. of glacial acetic acidwas added ml. of water, 6 grams of glycine, 3 grams of ammoniumpersulfate and 150 mg. of ferric chloride. The mixture was diluted toml. with water. Strips of No. Whatman filter paper (8 x 0.5 cm.) werepartially dipped in this solution and dried rapidly under a vacuum inthe absence of light. The impregnated portion was pale blue-white incolor but when it was contacted with a solution of uric acid or a serumor urine containing uric acid, a blue color developed, the intensity ofwhich was dependent upon the concentration of the uric acid present. Bycomparing the color of the strip with a standard color chart, twominutes after adding the solution, serum or urine, it was possible toestimate the uric acid content.

Example 3 TPTZ (260 mg.) was dissolved in 10 ml. of an aqueous solutioncontaining 18% acetic acid and 60 ml. of an aqueous solution containing10% glycine were added rapidly. To this mixture was added 15 ml. of anaqueous solution containing mg. of ferric chloride and 3 grams ofammonium persulfate. The resulting mixture was diluted to 100 ml. withwater and allowed to stand 2 hours. Strips of absorbent filter paperwere impregnated with this reagent solution by dipping and drying saidstrips in a current of warm air. The strips were used to estimate uricacid as described in the preceding example in sera and urine containingfrom 0 to 15 percent mg. uric acid.

In any of these examples the TPTZ can be replaced by an equal weight ofterpyridyl and essentially the same results obtained.

It has been found that when a persulfate is included in the uric acid,test compositions described herein, there is: no interference due to thepresence in the fluid being tested, of; glutathione, L-ergothioneine,cysteine, solicylates, gentisicacid, phenol, acetoacetate, iodide,cupric salts, citrate, lactate, pyruvate and phosphate.

What is claimed is: i

1. A composition for the detection of uric acid which comprises a ferricsalt and a substance selected from the group consisting of2,4,6-tri(2pyridyl)-1,3,5-triazine and 2,2':6',2"-terpyridyl, in a,buffered acidic medium.

2. A composition as claimed in claim 1 in which the ferric salt isferric chloride. 1

3. A composition as claimed in claim 1 which one part of ferric salt isemployedfor every two parts of 2,4,6-tri (2-pyridyl) -1,3,5-triazone or2,2':6',2"-terpyridyl.

4. A composition as claimed in claim 1 in which the buffer employed isselected from the group consisting of glycine-hydrochloric acid andglycine-acetic acid.

5. A composition as. claimed in claim 1 in which the butter exerts a pHof about 4.

6. A composition as claimed in claim 1 which further includes the use ofa persulfate.

7. A composition as claimed in claim 6 in which the persulfate isammonium persulfate.

8. A test device comprising a carrier member incorporating a compositionas claimed in claim 1.

9. A test device as claimed in claim 8 wherein the carrier member isbibulous paper.

References Cited- UNITED STATES PATENTS 1/1967 Bradbury et a1. 260296 D1/1971 Cottrell 23230 B MORRIS'YO. WOLK, Primary Examiner ROBERT M.REESE, Assistant Examiner

